Methods for Detecting Circulating Tumor Cells

Abstract

Nucleic acids and methods for amplifying and detecting mutant BRAF sequences are provided herein. In particular, nucleic acids and methods for amplifying and detecting BRAF sequences that encode the V600E BRAF mutant are provided herein.

1 . A method for determining whether a subject contains, in the peripheral circulation, a nucleic acid comprising a mutant BRAF gene or a fragment thereof, said method comprising:
 (a) providing nucleic acid from a peripheral blood sample obtained from said subject;   (b) contacting said nucleic acid with at least a first primer under conditions that will result, if a BRAF gene or fragment thereof is present in said peripheral blood sample, in amplification of said BRAF gene or fragment thereof, and   (c) determining whether said BRAF gene or fragment thereof contains a mutation as compared to a wild-type BRAF sequence.   
     
     
         2 . The method of  claim 1 , wherein said subject is a human. 
     
     
         3 . The method of  claim 1 , wherein said subject is diagnosed with cancer. 
     
     
         4 . The method of  claim 1 , wherein said nucleic acid is contained within cells in the peripheral circulation. 
     
     
         5 . The method of  claim 4 , wherein said cells are melanoma, papillary thyroid carcinoma, or colon cancer cells. 
     
     
         6 . The method of  claim 1 , wherein said peripheral blood sample is a serum sample. 
     
     
         7 . The method of  claim 1 , wherein said peripheral blood sample is a plasma sample. 
     
     
         8 . The method of  claim 1 , wherein said mutation is an adenine substitution for thymine at nucleotide 1799 relative to the adenine in the BRAF translation initiation codon. 
     
     
         9 . The method of  claim 1 , wherein said step (b) comprises contacting said nucleic acid with at least a first primer under conditions that will result, if said mutant BRAF gene or fragment thereof is present in said peripheral blood sample, in specific amplification of said mutant BRAF gene or fragment thereof, giving a first amplified product, and wherein said step (c) comprises detecting the presence or absence of said first amplified product, wherein the presence of said first amplified product indicates that said mutant BRAF gene or fragment thereof is present in said peripheral blood sample, and wherein the absence of said first amplified product indicates that said mutant BRAF gene or fragment thereof is not present in said peripheral blood sample. 
     
     
         10 . The method of  claim 9 , wherein said first primer is complementary to either strand of a wild-type BRAF nucleotide sequence, with the proviso that the nucleotide at the 3′ end of said first primer is not complementary to either strand of the wild-type BRAF nucleotide sequence. 
     
     
         11 . The method of  claim 9 , wherein said first primer has the sequence set forth in SEQ ID NO:3. 
     
     
         12 . The method of  claim 9 , wherein said detecting comprises gel electrophoresis, melting profile with an intercalating dye, hybridization with an internal probe, and/or real time PCR. 
     
     
         13 . The method of  claim 9 , wherein said first primer comprises a fluorescent label. 
     
     
         14 . The method of  claim 9 , further comprising
 (d) contacting said nucleic acid with at least a second primer under conditions that will result, if a non-mutant BRAF gene is present in said peripheral blood sample, in specific amplification of said non-mutant BRAF gene, giving a second amplified product; and   (e) detecting the presence or absence of said second amplified product.   
     
     
         15 . The method of  claim 14 , further comprising comparing the amounts of said first amplified product and said second amplified product. 
     
     
         16 . The method of  claim 15 , wherein said nucleic acid is contained within cells in the peripheral circulation, and wherein the relative levels of said first and second amplified products indicates the fraction of cells having said mutant BRAF gene in said peripheral blood sample. 
     
     
         17 . The method of  claim 14 , wherein said first primer has the nucleotide sequence set forth in SEQ ID NO:3, and wherein said second primer has the nucleotide sequence set forth in SEQ ID NO:5. 
     
     
         18 . The method of  claim 9 , further comprising, after step (a) and prior to step (b), contacting said nucleic acid with one or more degenerate primers under conditions that will result in universal amplification of said nucleic acid. 
     
     
         19 . An isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3. 
     
     
         20 . An isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:4. 
     
     
         21 . An article of manufacture comprising an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:3, and an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:4. 
     
     
         22 . The article of manufacture of  claim 21 , further comprising an isolated nucleic acid having the nucleotide sequence set forth in SEQ ID NO:5.